Human noroviruses (HuNoVs) are the leading cause of gastroenteritis worldwide causing 23 million cases of gastroenteritis annually and substantial economic impacts in the US alone. The lack of an animal disease model (until recently) and routine cell culture system for HuNoVs hinders knowledge of NoV replication strategies;consequently, no vaccines, anti-virals or therapies exist for this category B biodefense pathogen. Breakthroughs in enteric calicivirus [NoVs &sapoviruses (SaVs)] research that make it opportune to exploit these advances to propagate HuNoVs in vitro include: 1) Mediators in the intestinal micro-environment (intestinal content filtrates, bile acids) that promote in vitro replication of porcine SaV;2) Role of innate immunity in inhibiting murine NoV (MNV) infection and evidence for dendritic cells (DCs) and macrophages as in vivo and in vitro MNV targets;3) Histo-blood group antigens as potential receptors on human and pig enterocytes that influence susceptibility to HuNoV;4) Use of the dominant HuNoV outbreak strain (GII.4) with broad HBGA binding to develop gnotobiotic (Gn) pig and calf disease models for HuNoV;5) Preliminary, but unconfirmed data alleging HuNoV RNA replication in human three-dimensional (3-D) cell or organ cultures. Based on these key advances, our specific aims are: Aim 1. Develop ex vivo duodenal and jejunal organ cultures of Gn pig and calf intestine to propagate HuNoVs;Aim 2. Investigate use of 3-D cultures of pig (IPEC-J2) or human (HuTu80) small intestinal and other enterocyte cell lines to propagate HuNoVs;Aim 3. Establish primary dendritic cell (DC)/macrophage cultures from Gn pigs/calves and co-culture these with the homologous organ, 3-D or 2-D cultures, or co-culture the established DC/macrophage cell lines with 3-D or 2-D cultures to propagate HuNoV;Aim 4. Use in vivo animal-passaged or ex vivo organ- or 3-D culture-passaged HuNoVs and various conventional and non-conventional cell lines to propagate HuNoVs. We will test original and Gn pig and calf passaged HuNoV GII.4 strain or newly acquired HuNoVs in each of the 4 culture systems (Aims1- 4). We hypothesize that prior propagation in animals selects for HuNoV variants that adapt to cell culture, like we verified previously for in vitro adaptation of Gn pig-passaged human rotavirus. Medium supplements or conditions mimicking the intestinal microenvironment include intestinal content filtrates, bile acids and pancreatin that enhance propagation of fastidious enteric viruses, including enteric caliciviruses. Other innovative approaches include use of molecular beacons (monitor replication), growth factors (TGF2 and EGF) to promote epithelial cell growth and differentiation, use of HBGA matched organ and 3D cultures to reflect binding by selected HuNoV strains, co-culture strategies to mimic diversity of intestinal cell types including DCs/macrophages, and inhibition of innate immunity to enable in vitro replication of HuNoVs. Propagation of HuNoVs would be a major breakthrough to advance basic and translational studies of NoV replication leading to new strategies for their prevention and control and improved global public health. PUBLIC HEALTH RELEVANCE: Human noroviruses (HuNoVs) are emerging pathogens that infect individuals of all ages. They are the major cause of nonbacterial gastroenteritis worldwide causing 23 million cases of gastroenteritis leading to an estimated 50,000 hospitalizations and 500 deaths annually and substantial economic impacts in the US alone. Global outbreaks are increasing in number leading to increased morbidity in susceptible populations including travelers, those on cruise and military ships, military troops, hospital and nursing home patients, children in schools and day care centers and other settings where there are highly susceptible and changing populations in a confined space. Their low infectious dose, environmental resistance, strain diversity, shedding from asymptomatic individuals and varied transmission vehicles render HuNoVs highly contagious prompting their classification as category B biodefense pathogens by the NIAID, NIH. The lack of an animal disease model (until recently) and routine cell culture system for HuNoVs greatly hinders knowledge of NoV replication strategies;consequently, there are no vaccines, anti-viral drugs or therapies for their prevention and control. Severe and widespread outbreaks often necessitate the closing of schools, nursing homes and hospitals as the only means currently available for their control. We will exploit new advances in enteric calicivirus [NoVs and sapoviruses (SaVs)] research from our lab and others to develop methods to propagate HuNoVs in laboratory cell cultures. These include use of germfree pig and calf (only animal disease models for HuNoV) passaged HuNoV GII.4 strains that are dominant worldwide, use of culture medium supplements/conditions mimicking the intestinal microenvironment in which enteric caliciviruses grow, use of new types of intestinal organ and 3-D cell cultures and co-culture strategies mimicking the diversity of intestinal cell types, and the blocking of innate antiviral immunity in these cultures. Establishment of an in vitro cell culture system for HuNoVs will provide an in vitro platform to investigate the mechanism of HuNoV replication, to screen for anti- viral drugs and therapies, to serotype HuNoVs and to develop attenuated vaccines and evaluate the neutralizing antibody responses they elicit and correlates of protection. In vitro propagation of HuNoVs will provide the major breakthrough needed for development of new strategies and means to prevent and control HuNoV infections and thereby improve public health globally.